UV-VISIBLE SPECTROSCOPY INSTRUMENTATION.pptx

1. UV-VISIBLE SPECTROSCOPY
INSTRUMENTATION
Presented by: Dr Vijaya U. Barge
Vice Principal & Professor
Pune District Education Association’s
Shankarrao Ursal College of
Pharmaceutical Sciences & Research
Centre.

2. LEARNING OBJECTIVES:
After completing this sub-unit the students should be able:
To learn principle measurement of radiation.
To study about electronic transitions after absorption of
radiation.
To know the factors affecting spectral shift.
 To learn about theory of spectroscopy and Beer-Lambert law and
its deviations.
To study instrumentation used in UV-Visible spectroscopy.To study
components of UV-Visible spectrophotometer.
To learn about applications of UV-Visible spectroscopy.

3. INTRODUCTION
 Spectroscopy is the branch of Science that deals with the study of interaction of
electromagnetic radiation with matter.
 Spectrometer is an instrument design to measure the spectrum of a compound .
 Instrument used to measure the absorbance in UV (200 400nm) or Visible (400-
800nm) region is called UV Visible Spectrophotometer.
 A Spectrophotometer records the degree of absorption by a sample at different
wavelengths and the resulting plot of absorbance (A) versus wavelength (2) is known
as a Spectrum.

4. INSTRUMENTATION
 COMPONENTS OF
SPECTROPHOTOMETER
1) Source
2) Wavelength selectors or Dispersing
devices
3) Sample compartment
4) Detector
5) Recorder

5. LIGHT SOURCE
Requirements:
 It should be stable.
 It should provide continuous radiation .
 It must be of the sufficient intensity for the transmitted energy to be detected at the
end of the optical path.
TYPES OF LAMP :
1) HYDROGEN DISCHARGE LAMPS
2) DEUTERIUM LAMPS
3) TUNGSTEN LAMP
4) XENON DISCHARGE LAMP
5) MERCURY ARC LAMP

6. HYDROGEN DISCHARGE LAMP
 In these lamps a pair of electrodes is enclosed in a glass tube filled
with hydrogen gas under relatively high pressure.
 It is a continuous source.
 Covers a range 160-375nm.
 Stable, robust and widely used.
PROCESS
1) High voltage current is passed through the electrodes
2) Discharge of electrons occurs
3) Excitation of hydrogen molecules
4) Cause emission of UV radiation.

7. DEUTERIUM LAMPS
 Similar to hydrogen discharge lamp.
 Deuterium is filled in place of hydrogen.
 The intensity of radiation emitted is 3 to 5
times the intensity of a hydrogen lamp.
 More expensive than hydrogen lamp.
 Used when high intensity is required.

8. TUNGSTEN LAMP
 Similar in its functioning to an electric bulb.
 It provides a supply of radiation in the wavelength
range of 320-2500nm.
 Continuous source of light.
 When tungsten filament is heated to
incandescence by an electric current ,the light is
produced.
 The glass bulb enclosing the filament contains a
low pressure of inert gas, usually argon.
 Small amounts of halogen like iodine is added to
improve the intensity (Tungsten-Iodine lamp).

9. XENON DISCHARGE LAMP
 Xenon gas is stored in lamps at 10-30 atm pressure.
 It contain 2 tungsten electrodes that are separated by a distance of about 8mm.
 When current passes through xenon cause thermal excitation.
 It produces greater UV radiation than the hydrogen lamp.
 Continuous source.

10. MERCURY ARC LAMP
 In this mercury vapour is stored under high
pressure and the excitation of mercury
atoms is done by electric discharge.
 Mercury vapour at high pressure give
intense lines on continuous background
above 350nm.
 Low pressure mercury vapour gives an
additional line at 254nm.

11. WAVELENGTH SELECTOR
 Converts polychromatic light to monochromatic
light.
 Filters and Monochromators are used for this
purposes.
FILTERS :
 It is frequently necessary to filter or remove wide
bands of radiation from a signal.
 Filters isolate a wider band than the
monochromators.

12. MONOCHROMATORS
 It is used to disperse the heterochromic radiation into its component
wavelength and to permit the isolation of desired portion of the spectrum
.
 It consists of an entrance slit, an exit slit and a dispersing device either a
prism or grating.
 Materials of construction should be selected withcare to suit the range in
which it has to work.
For eg :
 Quartz for ultraviolet.
 Normal glass for visual range.
 Alkali halides for IR region.
 Gratings are cheaper than prism.

13. PRISM
 Made up of glass , quartz or fused silica.
 Quartz or fused silica is the choice of material
of UV spectrum.
 When white light is passed through the glass
prism, dispersion of polychromatic light in
rainbow occurs .
 Now by the rotation of the prism different
wavelengths of the spectrum can be made to
pass through in exit slit on the sample.
 The effective wavelength depends on the
dispersive power of prism material and the
optical angle of the prism.

14. GRATING MONOCHROMATOR
 Gratings are of 2 types
1. Diffraction grating
2. Transmission grating

15. DIFFRACTION GRATING
 More refined dispersion of light is obtained by means of diffraction gratings.
 These consists of large no.of parallel lines(grooves) about 15000-30000/inch is
ruled on highly polished surface of aluminum.
 To make the surface reflective, a deposit of aluminum is made on the surface.
In order to minimize to greater amounts of scattered radiation and appearance
of unwanted radiation of other spectral orders, the gratings are blazed to
concentrate the radiation into a single order.
TRANSMISSION GRATING
 It is similar to diffraction grating but refraction takes place instead of
reflection.
 Refraction produces reinforcement.
 This occurs when radiation transmitted through grating reinforces with the
partially refracted radiation.

16. SAMPLE HOLDER/ CUVETTES
 The cells or cuvettes are used for handling liquid samples.
 The cell may either be rectangular or cylindrical in nature.
 For study in UV region the cells are prepared from quartz or
fused silica whereas colour corrected fused glass is used for
visible region.
 Cleaning is carried out washing with distilled water or with
dilute alcohol, acetone.
 The surfaces of absorption cells must be kept scrupulously
clean.
 No fingerprints or blotches should be present on cells.

17. DETECTORS
 Device which converts light energy into electrical signals,
that are displayed on readout devices.
 The transmitted radiation falls on the detector which
determines the intensity of radiation absorbed by sample.
 The followed types of detectors are employed in
instrumentation of absorption spectrophotometer.
1) Barrier layer cell/Photovoltaic cell.
2) Phototubes/Photo emissive tube.
3) Photomultiplier tube.

18. BARRIER LAYER CELL/ PHOTOVOLTAIC CELL
 The detector has a thin film metallic layer coated with
silver or gold and act as another electrode.
 It also has a metal base plate which act as another
electrode.
 These 2 layers are separated by a semiconductor layer of
selenium.
 This creates a potential difference between two
electrodes & causes the flow of current.
 When it is connected to galvanometer, a flow of current
observed which is proportional to the intensity and
wavelength of light falling on it.
 When light radiation falls on selenium layer, electrons
become mobile and are taken up by transparent metal
layer.

19. PHOTOTUBES/PHOTOEMISSIVE
TUBES
 Consists of a evacuated glass tube with
a photocathode and collector anode.
 The surface of photocathode is coated
with a layer of elements like cesium,
silver oxide or mixture of them.
 When radiant energy falls on
photosensitive cathode, electrons are
attracted to anode causing current to
flow.
 More sensitive compared to barrier
layer cell and therefore widely used.

20. INSTRUMENT DESIGN
SINGLE-BEAM SPECTROPHOTOMETER
 A beam of radiation pass through a single cell, the reference cell is used to set the
absorbance scale at zero for the wavelength to be studied.
 It is then replaced by sample cell to determine the absorbance of the sample at that
wavelength.
 This was earliest design and is still use in both teaching and industrial labs.
 It requires a stabilized voltage supply to avoid errors resulting from changes in the beam
intensity.

21. DOUBLE-BEAM SPECTROPHOTOMETER
DOUBLE-BEAM-IN-SPACE INSTRUMENT
 Two beams are formed in space by a V-shaped mirror called beam splitter.
 One beam passes through the reference solution to a photodetector, and the 2nd
simultaneously traverses the sample to a 2nd matched detector.
 The 2 outputs are amplified, and their ratio or logarithm of their ratio is determined
electronically or by a computer and displayed by the readout device.

22. DOUBLE-BEAM-IN-TIME
INSTRUMENT
 The beams are separated in time by a rotating sector mirror that
directs the entire beam from the monochromator first through the
reference cell and then through the sample cell.
 The pulses of radiation are recombined by another sector mirror,
which transmits one pulse to other to the transducer.
 The motor-driven sector mirror is made up of pie shape segments,
half of which are mirrored and half of which are transparent.
 The mirrored sections are held in place by blackened metal frames
that periodically interrupt the beam and prevent its reaching the
transducer.
 The double-beam-in-time approach is generally preferred because of
the difficulty in matching the 2 detectors needed for the double-
beam-in-space design.

24. REFERENCE
 Dr Sanjay G. Walode , Sr Chandan R.S , Instrumental Methods Of
Analysis , First Edition , September 2020.
 Willard , Merrit , Dean , Settle , Instrumental Method Of Analysis ,
7th edition , CBS publishers & Distributors Pvt. Ltd.
 Gurdeep R . Chatwal , Sham K Anand , Instrumental Method Of
Chemical Analysis.
 Douglas A Skoog , Donald M West , F . James Holler , Skoog & West's
Fundamental of Analytical Chemistry , Cengage Technology Edition.